Summary The more than ~25,000 human genes give rise to many more proteins, a.o. by alternative splicing. Genome-wide approaches using database annotations attempt to reveal patterns of protein interactions, however generally ignore the protein locations. Mass spectrometry handles a huge number of proteins ranging widely in abundance, yet its resolution can be enhanced by analyzing purified organellar fractions. This proposal aims to generate proteome inventories of plasma membranes (PM) and endosomes in a disease-related context. Classical purification schemes cannot be used as disease-related mutations often alter the physical parameters used for organellar isolation. This caveat is circumvented by administering surface-coated superparamagnetic iron oxide nanoparticles (SPIONs). We have developed lipid-coated SPIONs which target the cell surface and allow purification of PM. By exploring the PM proteome we will search for stage-specific or unique expression of channels, receptors or adhesion molecules that may become novel targets or early biomarkers in disease. SPION variants will further be optimized to load endosomes. Coupling biomolecules (ligands, toxins, drugs) combined with pulse-chase experiments will allow isolating distinct endosome populations. Many diseases including neurodegenerative and metabolic diseases originate from a block in endosomal trafficking and degradation although the causal genes are not always identified. By comparing the endosomal proteomes in patient cell lines with control, we aim to identify aberrant protein expression patterns from which causal genes and/or novel biomarkers could be identified. Production and QC of SPIONs is done at IMEC and VIB11. Mass spectrometry driven proteomics to catalogue organellar proteomes is performed at VIB9.